GENE EXPRESSION ANALYSIS METHOD USING TWO DIMENSIONAL cDNA LIBRARY

Abstract

The present invention provides a method and/or means for collecting and analyzing an individual cell in a tissue, and at the same time, quantitatively monitoring the expression levels of various genes while keeping two-dimensional information in the tissue. Specifically, the present invention provides a method comprising preparing a cDNA library from mRNA while keeping two-dimensional cellular distribution information and obtaining the gene expression levels at any site or all sites at a level of single cell. More specifically, the present invention provides a method comprising preparing a cDNA library in a sheet-form from mRNA while keeping two-dimensional cellular distribution information and repeatedly using the cDNA library in the detection of the gene expression, thereby allowing measurement of the expression distribution for a number of genes at a high accuracy.

1 . A method for analyzing a gene expression profile, comprising the steps of:
 (a) hybridizing a test nucleic acid in a sample with a nucleic acid probe which has been two-dimensionally distributed and immobilized onto a support;   (b) synthesizing cDNA having a complementary sequence to the sequence of the test nucleic acid and preparing a two-dimensional cDNA library on the support; and   (c) detecting gene expression in the sample by using the two-dimensional cDNA library.   
     
     
         2 . The method according to  claim 1 , wherein the sample contains a plurality of cells and the gene expression profile is analyzed for each of the cells. 
     
     
         3 . The method according to  claim 1 , wherein the sample contains a plurality of cells, the gene expression profile is analyzed for each of the cells and the gene expression profiles of cells are compared. 
     
     
         4 . The method according to  claim 1 , wherein the sample is a biological tissue sample, and a two-dimensional cDNA library is prepared while keeping two-dimensional positional information of cells contained in the biological tissue sample. 
     
     
         5 . The method according to  claim 1 , wherein the sample is a biological tissue sample and a two-dimensional cDNA library is prepared while keeping two-dimensional positional information within cells contained in the biological tissue sample. 
     
     
         6 . The method according to  claim 4 , wherein the biological tissue sample is a tissue section sample and a test nucleic acid in the tissue section sample is transferred to the support to hybridize with the nucleic acid probe. 
     
     
         7 . The method according to  claim 6 , wherein the transfer of the test nucleic acid to the support is performed by electrophoresis. 
     
     
         8 . The method according to  claim 1 , wherein the sample is an array of cells two-dimensionally held. 
     
     
         9 . The method according to  claim 1 , wherein the test nucleic acid is selected from the group consisting of messenger RNA (mRNA), non-coding RNA (ncRNA) and DNA, and fragments thereof. 
     
     
         10 . The method according to  claim 9 , wherein the test nucleic acid is mRNA and the nucleic acid probe is a DNA probe containing a poly-T sequence. 
     
     
         11 . The method according to  claim 1 , wherein the support is at least one selected from the group consisting of a sheet, a membrane, a gel thin film, a capillary plate and beads. 
     
     
         12 . The method according to  claim 1 , wherein the support has a plurality of micro spaces two-dimensionally segmented. 
     
     
         13 . The method according to  claim 12 , wherein the interval between the micro spaces is smaller than the size of a cell. 
     
     
         14 . The method according to  claim 12 , wherein the two-dimensional cDNA library retains cDNAs in a plurality of micro spaces two-dimensionally segmented in the support. 
     
     
         15 . The method according to  claim 1 , further comprising transferring the cDNAs retained in the two-dimensional cDNA library to a second support to hybridize with a nucleic acid probe which has a complementary sequence to the cDNA and a fragment thereof and has been two-dimensionally distributed and immobilized onto the second support, thereby preparing a second two-dimensional cDNA library. 
     
     
         16 . The method according to  claim 1 , further comprising generating a nucleic acid fragment corresponding to cDNA retained in the two-dimensional cDNA library, transferring the nucleic acid fragment to the second support while keeping the two-dimensional positional information of the two-dimensional cDNA library, thereby preparing a second two-dimensional cDNA library. 
     
     
         17 . The method according to  claim 16 , wherein the nucleic acid fragment corresponding to cDNA contains a complementary sequence of the cDNA. 
     
     
         18 . The method according to  claim 16 , wherein the nucleic acid fragment corresponding to cDNA contains a complementary sequence of cDNA and a known sequence unrelated to the cDNA sequence. 
     
     
         19 . The method according to  claim 1 , wherein a labeled nucleic acid probe specific to the gene to be detected is hybridized with cDNA retained in the two-dimensional cDNA library and gene expression is detected based on the label. 
     
     
         20 . The method according to  claim 19 , wherein the label is a fluorescent label or a chemiluminescent label. 
     
     
         21 . The method according to  claim 1 , wherein a nucleic acid probe specific to the gene to be detected is hybridized with cDNA retained in the two-dimensional cDNA library; a nucleic acid sequence contained in the nucleic acid probe is subjected to an amplification reaction; and gene expression is detected based on an amplified product. 
     
     
         22 . The method according to  claim 21 , wherein the amplification reaction is a polymerase chain reaction (PCR). 
     
     
         23 . The method according to  claim 1 , wherein a nucleic acid padlock probe specific to the gene to be detected is hybridized with cDNA retained in the two-dimensional cDNA library; the padlock probe hybridized is subjected to a ligation reaction to form a ring form probe; a rolling circle amplification (RCA) reaction is performed with the ring form probe as a template; and gene expression is detected based on an amplified product. 
     
     
         24 . The method according to  claim 21 , wherein the amplified product is detected by use of fluorescence or chemiluminescence. 
     
     
         25 . The method according to  claim 1 , wherein the two-dimensional cDNA library is repeatedly used to perform the step of detecting gene expression. 
     
     
         26 . The method according to  claim 1 , further comprising comparing the results from the detection of gene expression in the two-dimensional cDNA library with two-dimensional positional information of the sample to obtain correlation data between a specific position in the sample and gene expression. 
     
     
         27 . A method for analyzing a profile of a molecule contained in a sample, comprising the steps of:
 (a) binding a test molecule in a sample to an antibody or aptamer which has been two-dimensionally distributed and immobilized onto a support;   (b) forming a ring probe specific to binding of the test molecule to the antibody or aptamer, by use of a proximity ligation method;   (c) preparing a two-dimensional cDNA library by using the ring probe or a nucleic acid fragment generated with the ring probe as a template; and   (d) detecting the presence of a molecule in the sample by using the two-dimensional cDNA library.   
     
     
         28 . The method according to  claim 27 , wherein the molecule is a protein.